The molecular mechanisms involved in synapse formation in the central nervous system are unclear. Although it is known that contact of the presynaptic axon with the postsynaptic dendrite induces pre-and post-synaptic differentiation of the synapse, the molecules involved in this process have not been identified. Studies of the neuromuscular junction have shown that presynaptic release of the proteoglycan agrin induces postsynaptic differentiation and clustering of the postsynaptic nicotinic acetylcholine receptor. To investigate the factors involved in the postsynaptic differentiation of excitatory synapses in the CNS we have recently reconstituted synapse formation between axons and non-neuronal cells expressing glutamate receptors and other synaptic components. Incubation of neurons with non-neuronal cells under these conditions promotes differentiation of the presynaptic nerve terminal and clustering of AMPA receptors at the point of contact between the axon and the transfected non-neuronal cell. Electrophysiological recordings of the non-neuronal cells remarkably show synaptic excitatory postsynaptic currents (EPSCs) and mEPSCs. Using this system we will characterize the requirements for postsynaptic clustering of AMPA receptors. Initially we will characterize the structural domains of the AMPA receptor subunits required for this clustering. Deletions of the intracellular C-terminal region of the subunits will be performed to investigate whether intracellular protein interactions are required. Deletions of extracellular domains of the subunits will be performed to see if direct interaction of the AMPA receptor subunits with proteins secreted from the axon is required for receptor clustering. Information from these studies will be used for isolation of the molecules that promote AMPA receptor clustering. These studies will help identify molecules involved in synapse formation and modulation during development and in the adult. Since many neurological and psychiatric diseases result from defects in synaptic transmission, understanding the basic mechanisms regulating synaptic function is critical for the development of therapeutic treatments for these disorders.